The Retinoblastoma (pRB) pathway is a key tumor-suppressor pathway. It plays a pivotal role in normal development while its functional inactivation occurs in most cancers. The family of E2F factors is one of the key downstream targets of pRB and deregulated E2F activity is thought to drive proliferation in tumor cells. In contrast, less is known about novel Hippo tumor-suppressor pathway. However, recent studies in flies and in mammals suggested that Hippo pathway controls tissue homeostasis while its deregulation leads to tumorigenesis, thus, underscoring its important role in influencing the malignant state. Using a Drosophila model system, we have discovered that Hippo pathway strongly cooperates with pRB pathway in limiting cell proliferation and in maintenance of the state of terminal differentiation. Combined inactivation of both pathways gives rise to inappropriate proliferation and, surprisingly, to extensive dedifferentiation. Importantly, these two facets of a crosstalk are independent of each other and reflect distinct functions of the pRB pathway. Experiments in this proposal will use a combination of approaches and newly developed tools to define the cellular mechanisms underlying cooperation between the two pathways and to test the importance of these mechanisms in vivo. In Aim 1, we will decipher an E2F-dependent aspect of cooperation between Hippo and RB pathways in limiting cell proliferation. In Aim 2 we will investigate how the two pathways are integrated to maintain the differentiated state. This knowledge could eventually form a basis for manipulating the pathways and may help in designing new anticancer therapies for treating cancers. PUBLIC HEALTH RELEVANCE: The focus of proposed research is to investigate the mechanisms underlying cooperation between the pRB pathway and a novel Hippo tumor suppressor pathway in control of cell proliferation and maintenance of the differentiated state in vivo. Notably, we have discovered that combined inactivation of both pathways leads to dramatic dedifferentiation. Therefore the results of the proposed research may help in designing of efficient anticancer therapies particularly those based on differentiation-induced agents.